A DCL3 dicing code within Pol IV-RDR2 transcripts diversifies the siRNA pool guiding RNA-directed DNA methylation

Abstract

In plants, selfish genetic elements including retrotransposons and DNA viruses are transcriptionally silenced by RNA-directed DNA methylation. Guiding the process are short interfering RNAs (siRNAs) cut by DICER-LIKE 3 (DCL3) from double-stranded precursors of ~30 bp that are synthesized by NUCLEAR RNA POLYMERASE IV (Pol IV) and RNA-DEPENDENT RNA POLYMERASE 2 (RDR2). We show that Pol IV's choice of initiating nucleotide, RDR2's initiation 1-2 nt internal to Pol IV transcript ends and RDR2's terminal transferase activity collectively yield a code that influences which precursor end is diced and whether 24 or 23 nt siRNAs are produced. By diversifying the size, sequence, and strand specificity of siRNAs derived from a given precursor, alternative patterns of DCL3 dicing allow for maximal siRNA coverage at methylated target loci.

The mechanisms of siRNA selection by plant Argonaute proteins triggering DNA methylation

“…Previous reports showed that DCL3 prefers to cleave dsRNA precursors from the end with 5′ A and/or unstable sequence (23)(24)(25). Thus, we cannot exclude the possibility that the siRNAs with the 5′ A and the thermodynamically unstable ends are enriched at the DCL3-mediated cleavage steps but not at the loading step.…”

“…Sequencing of total small RNAs in plant cells showed that about 60% of 24-nt siRNAs have a 5′ A (18). This enrichment of 24A siRNAs can be explained by the biased selection of transcription start site (TSS) by Pol IV and the substrate specificity of DCL3: Pol IV TSSs exhibit preference for A or G at the +1 position (21), and DCL3 prefers to cleave dsRNA precursors from the end with 5′ A and/or unstable terminus (22)(23)(24)(25). On the other hand, sequencing of small RNAs loaded into AGO4/6 showed a slightly higher percentage of 24A siRNAs than total small RNAs (18), suggesting that 24A siRNAs are further enriched in the loading step.…”

Enzymatic reactions of AGO4 in RNA-directed DNA methylation: siRNA duplex loading, passenger strand elimination, target RNA slicing and sliced target retention

“…These enzymes physically associate (Haag et al, 2012) and carry out coupled reactions that transcribe target locus DNA sequences into double-stranded RNAs of ~25-40bp (Fukudome et al ., 2021; Huang et al, 2021; Singh et al, 2019). DICER-LIKE 3 (DCL3) then cuts the dsRNAs into siRNAs, guided by sequence and structural features at the ends of precursor duplexes (Loffer et al, 2022). Resulting dicing products consist of a 24 nt siRNA strand paired with either a 23 nt strand (24/23 duplexes) or a 24 nt strand (24/24 duplexes) (Loffer et al ., 2022; Nagano et al, 2014), one strand of which ultimately becomes stably associated with ARGONAUTE 4 (AGO4).…”

“…DICER-LIKE 3 (DCL3) then cuts the dsRNAs into siRNAs, guided by sequence and structural features at the ends of precursor duplexes (Loffer et al, 2022). Resulting dicing products consist of a 24 nt siRNA strand paired with either a 23 nt strand (24/23 duplexes) or a 24 nt strand (24/24 duplexes) (Loffer et al ., 2022; Nagano et al, 2014), one strand of which ultimately becomes stably associated with ARGONAUTE 4 (AGO4). The siRNAs are then thought to guide AGO4 to RdDM target loci by basepairing to transcripts generated by NUCLEAR RNA POLYMERASE V (Pol V) (Liu et al, 2018; Wierzbicki et al, 2008; Wierzbicki et al, 2009), with protein-protein interactions between AGO4 and the Pol V largest subunit (NRPE1), and/or the Pol V-associated protein, SPT5L also implicated in target locus interactions (Bies-Etheve et al, 2009; El-Shami et al, 2007; Li et al, 2006).…”

“…Although DCL3 dicing frequently generates duplexes consisting of 23 nt siRNAs basepaired with 24 nt siRNAs (Loffer et al ., 2022), siRNAs that co-immunoprecipitate with AGO4 are almost exclusively 24 nt (Havecker et al, 2010; Qi et al, 2006; Wang et al, 2011). Thus, the fates and functions of 23 nt siRNAs is unclear.…”